The oligosaccharide moieties of glycoconjugates play important roles in several biological processes of a cell, including the folding and transport of glycoproteins across cellular compartments. For the biosynthesis of these complex oligosaccharides intricate machineries exists in a cell. Defective glycan synthesis has serious pathological consequences and results in several human diseases. The oligosaccharide moieties bind to cellular proteins with high specificity and modulate the homo- and hetro-dimerization of glycoproteins. Due to the conformational flexibility of oligosaccharides, the torsional angles of a disaccharide unit, especially around the1-6-linkage, adjust in such a way that the side groups of the oligosaccharides orient themselves in a manner that promotes favorable interactions with the binding residues of the protein. Branched oligosaccharides cross-link proteins and generate infinite networks of protein-carbohydrate complexes, resulting in the modulation of various cell responses. Although all beta-4Gal-T-family members, that are responsible for the synthesis of beta-linked Gal moieties in different oligosaccharides, transfer Gal to GlcNAc, each recognizes differently the remaining monosaccharide units of the oligosaccharide to which GlcNAc is attached. The sequence comparison of the human b4Gal-T family members and the structural homology models based on the 3D structure of b4Gal-T1 reveals only a little or no variation in the GlcNAc binding site among the family members, where as the extended oligosaccharide binding region shows significant variations. This indicates that these enzymes may prefer different GlcNAc containing oligosaccharides as their preferred sugar acceptors. To determine the exact mode of binding of the oligosaccharide in the binding site we have carried out the crystal structure analysis of the b4Gal-T1-oligosaccharide complexes, enzyme kinetic analysis and MD simulations. Defining the oligosaccharide binding site of b4Gal-T1 by crystal structure investigations of the complexes with the oligosaccharides : By molecular modeling and docking studies we have previously defined the oligosaccharide binding site of b4Gal-T1, the 3D-structure of which has been determined in our laboratory, either in complex with UDP-galactose and Mn2+ion, or in complex with alpha-lactalbumin and N-acetylglucosamine (see Project # Z01 BC 009304), or of the mutant Met344His-b4Gal-T1 in complex with chitobiose (see Project # Z01 BC 009305). Examination of the GlcNAc binding site in b4Gal-T1 from the crystal structure reveals an "open canal shaped" extended sugar binding site that lay behind the GlcNAc binding site. This site is formed by the residues from three regions; residues 280 to 289, residues 319 to 325 and residues 359 to 368. In the crystal structure of b4Gal-T1-LA-complex, LA binds to this region and therefore LA is expected to compete with the GlcNAc containing oligosaccharides, such as chitobiose. Crystallization of the wild type b4Gal-T1with the acceptor either in the presence or absence of UDP has not been successful. This is mainly due to the absence of the acceptor binding-site in the apo-b4Gal-T1 that exists in the open conformation. The enzyme has been crystallized in the closed conformation, where the acceptor site is present, only when UDP-Gal is bound. Although UDP or the acceptor molecules can induce the essential conformational changes, such complexes have been crystallized thus far only in the presence of LA. Since LA binds to the extended sugar binding site it is not possible to crystallize b4Gal-T1 with the oligosaccharide acceptors in the presence of LA. In a previous study we showed (see Project # Z01 BC 009305) that when residue Met344 in bovine b4Gal-T1 is mutated to histidine, the mutant M344H in the presence of Mn2+ and UDP-hexanolamine readily changes to the closed conformation that creates the acceptor binding site, thereby facilitating the structural analysis of the enzyme with various oligosaccharide acceptors. The branch specificity of human b4-Gal-T1 was investigated with the mutant human Met344His-b4Gal-T1 that was crystallized in complex with UDP-hexanolamine, Mn2+, and different trisaccharides. The following trisaccharides of the N-Glycan moiety were used: GlcNAcb1-2Mana1-6Man (1-6-arm), GlcNAc b1-2Mana1-2Man (1-2-arm), GlcNAcb1-4Mana1-3Man (triantennary), GlcNAcb1-2Mana1-3Man (1-3-arm), and GlcNAcb1-4GlcNAcb1-4GlcNAc (chitotriose). Crystal data was collected at 2.0 for all the complexes, except for the complex with triantennary trisaccharide (1.9 ). In all the structures the mutant human b4Gal-T1-Met344His was found to be in the closed conformation with the trisaccharides bound to it. The electron density for the core Man residue (the third residue from the non-reducing end) among the trisaccharides bound to b4Gal-T1 was clearly observed in the 1-6-arm trisaccharide where the core mannose rests over Tyr286 making hydrophobic interactions. In contrast, the core Man residue of the 1-3-arm trisaccharide possesses vague electron density with partial occupancy. The enzyme kinetic analysis also indicated a preferential binding of the 1-6-arm trisaccharide to the mutant enzyme. The Km of GlcNAcb1-2Mana1-6Man is approximately 10-fold lower than the Km for GlcNAcb1-2Mana1-3Man and GlcNAcb1-4Mana1-3Man, and 22-fold lower than the Km for GlcNAc b1-2Mana1-2Man and chitotriose. However, the catalytic activity of the Met344His mutant, in the presence of Mn2+, with the trisaccharide GlcNAcb1,2Mana1-6Man was inhibited above 0.1 mM concentrations, whereas much higher concentrations (1-2 mM) for the other four trisaccharides where needed to inhibit the catalytic activity. Since b4Gal-T1 binds strongly to GlcNAcb1-2Mana1-6Man and the turnover number, kcat, is hardly reduced, the catalytic efficiency (kcat/Km) of the enzyme with this trisaccharide is high compared to the other trisaccharides used in this study. Based on the structural and kinetic analysis it is proposed that b4Gal-T1 may prefer to transfer Gal to 1-3-arm in a bi- or tri- or tetra-antennary oligosaccharide while as it may act as lectin when concentration of glycan with 1-6 arm is higher than the glycan with 1-3 arm.